Phosphonic acids represent a potent, yet underexploited, group of bioactive compounds with great promise in the treatment of human disease. A wide variety of phosphonates are produced in nature and many have useful bioactive properties. Among these are fosfomycin, a common treatment for acute cystitis during pregnancy, and FR900098, a novel treatment (and cure!) for malaria. Others include phosphinothricintripeptide (PIT), a widely used herbicide, and K-26, a compound with great promise in the treatment of high blood pressure. The targets of phosphonate inhibitors vary widely, allowing their use in the treatment of a wide variety of conditions, yet despite their utility, surprisingly little is known regarding the biosynthesis of these natural products. As outlined in this proposal, we believe there is ample reason to believe that (i) additional natural phosphonic acid products remain to be discovered (including ones with novel therapeutic targets), (ii) that a wealth of novel biochemistry and secondary metabolism will be discovered during characterization of phosphonic acid biosynthetic pathways, (iii) that currently known compounds can be modified and improved and (iv) that more economical methods for their production can be developed. The proposed Program Project addresses each of these topics via a multidisciplinary research program involving microbiology, biochemistry, chemistry, metabolic engineering and structural biology. We propose four intertwined research projects to discover, design and develop novel and known phosphonic acid antibiotics. The first project involves discovery, identification and characterization of gene clusters encoding phosphonic acid biosynthesis using microbial genetics and molecular biology. The second project involves the chemical and biochemical characterization of these biosynthetic pathways, including several entirely unprecedented enzymatic reactions involved in synthesis of these C-P bond-containing compounds. The third project involves bioengineering of natural and designed phosphonic acid biosynthetic pathways to allow overproduction of phosphonic acid antibiotics. The fourth project is focused on the development and use of mass-spectrometry to detect and quantify phosphonic acids and to characterize the biochemical reactions involved in their synthesis. The project will be housed in the new Institute for Genomic Biology at the University of Illinois, where the program project team will occupy a single, large contiguous laboratory.